Human DNases Research Articles

'Above all, do no harm': safeguarding pluripotent stem cell therapy against iatrogenic tumorigenesis.

Human pluripotent stem cells are the foundations of regenerative medicine. However, the worst possible complication of using pluripotent stem cells in therapy could be iatrogenic cancerogenesis. Nevertheless, despite the rapid progress in the development of new techniques for induction of pluripotency and for directed differentiation, risks of cancerogenic transformation of therapeutically implanted pluripotent stem cells still persist. 'Above all, do no harm', as quoted from the Hippocratic Oath, is our ultimate creed. Therefore, the primary goal in designing any therapeutic regimes involving stem cells should be the elimination of any possibilities of their neoplasmic transformation. I review here the basic strategies that have been designed to attain this goal: sorting out undifferentiated, pluripotent stem cells with antibodies targeting surface-displayed biomarkers; sorting in differentiating cells, which express recombinant proteins as reporters; killing undifferentiated stem cells with toxic antibodies or antibody-guided toxins; eliminating undifferentiated stem cells with cytotoxic drugs; making potentially tumorigenic stem cells sensitive to pro-drugs by transformation with suicide-inducing genes; eradication of differentiation-refractive stem cells by self-triggered transgenic expression of human recombinant DNases. Every pluripotent undifferentiated stem cell poses a risk of neoplasmic transformation. Therefore, the aforementioned or other novel strategies that would safeguard against iatrogenic transformation of these stem cells should be considered for incorporation into every stem cell therapy trial.

Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2, DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death.

IntroductionThe worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors.Specific aimThe specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases).MethodsThe DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers’ bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies’ guided DNA vectors delivered the transgenes for the human recombinant DNases’ into proliferating stem cells.ResultsDifferentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases resulted in complete collapse of the chromatin architecture and degradation of the proliferating cells’ genomic DNA. The proliferating stem cells, but not the differentiating ones, were effectively induced to die.ConclusionHerein, we describe attaining the proof-of-concept for the strategy, whereby transgenic expression of the genetically engineered human recombinant DNases in proliferating and directed differentiation resisting stem cells leads to their death. This novel strategy reduces the risk of iatrogenic neoplasms in stem cell therapy.

Two N-Linked Glycosylation Sites (Asn18 and Asn106) Are Both Required for Full Enzymatic Activity, Thermal Stability, and Resistance to Proteolysis in Mammalian Deoxyribonuclease I

Deoxyribonuclease I (DNase I) is known to be a glycoprotein, and two potential N-linked glycosylation sites (N18 and N106) are known for mammalian enzymes. In the present study, N18 and N106 were mutated in order to investigate the biological role of N-linked glycosylation in three mammalian (human, bovine, and equine) DNases I. The enzyme activities of N18Q and N106Q were lower than that of the wild type, and that of the double mutant (N18Q/N106Q) was lower than those of the single mutants, in accord with the sugar moiety contents in the three mammals. In addition, all mutant enzymes were unstable to heat, suggesting that both sites are required for heat stability. Moreover, in human and equine enzymes, the N18Q and N106Q mutant enzymes were less resistant to trypsin, while N18Q/N106Q was the most sensitive to trypsin. As for bovine DNase I, the trypsin resistance of N18Q and N106Q was similar to that of the wild type, but that of N18Q/N106Q decreased in a time-dependent manner. On the other hand, N-linked glycosylation was not related to pH sensitivity. The results of the present study suggest that N18 and N106 are both necessary for (i) full enzymatic activity, (ii) heat-stability, and (iii) trypsin resistance.

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me 2+) ions, with the level of activity decreasing in the order Mn 2+ ≥ Mg 2+ > Ca 2+ ≥ Cu 2+ > Co 2+ ≥ Ni 2+ ≥ Zn 2+, whereas Fe 2+ did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me 2+ ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me 2+ ions in the 0.1–12 mM range, depending on the particular metal ion. In the presence of all Me 2+ ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu 2+, Zn 2+, or Ca 2+ inhibited the Mg 2+-dependent hydrolysis of scDNA, while Ni 2+, Co 2+, and Mn 2+ activated this reaction. The Mn 2+-dependent hydrolysis of scDNA was activated by Ca 2+, Ni 2+, Co 2+, and Mg 2+ ions but was inhibited by Cu 2+ and Zn 2+. After addition of the second metal ion, only in the case of Mg 2+ and Ca 2+ or Mn 2+ ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0–100%) in the presence of Mn 2+, Ca 2+, and Mg 2+ ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn 2+, Ca 2+, and Mg 2+ ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.

Comparison of Enzymatic Properties of DNA‐Abzymes and Human DNAses

AbstractFor Abstract see ChemInform Abstract in Full Text.

Comparison of Enzymatic Properties of DNA‐Abzymes and Human DNAses

DNA‐hydrolyzing antibodies (DNA‐abzymes, Abz) were shown to be good biochemical markers of some autoimmune diseases such as systemic lupus erythematosus (SLE) and multiple sclerosis (MS). To better understand mechanisms of abzyme generation, one needs to know optimal conditions for DNA hydrolysis by DNA‐abzymes, as well as their enzymatic properties in comparison with those of enzymes possessing the same activity. In contrast to human urine deoxyribonucleases, DNA‐hydrolyzing antibodies efficiently digested both single‐ and double‐strand DNA. It was shown that polyclonal antibodies (Abs) in MS may contain up to several types of DNase activities, either activated by metal ions or not.

Lactoferrin Is the Major Deoxyribonuclease of Human Milk

Lactoferrin is the major iron-transferring protein of human barrier fluids such as blood and milk. It is a polyfunctional protein capable of binding DNA exposed on the surface of various cells. Electrophoretically homogenous lactoferrin was prepared by sequential chromatography of human milk proteins on DEAE-cellulose, heparin-Sepharose, and Sepharose containing immobilized anti-lactoferrin antibodies. By subsequent chromatography on Blue Sepharose the resulting lactoferrin was fractionated into several subfractions with different affinity for the sorbent, and this was associated with separation of additional lactoferrin peaks with DNase activity from the main peak. By various techniques, in particular, by in situ testing the DNase activity of lactoferrin in a DNA-containing gel after SDS-electrophoresis, hydrolysis of DNA was for the first time shown to be an intrinsic property of lactoferrin. The substrate specificity of lactoferrin in hydrolysis of DNA was different from specificities of known human DNases. Hydrolysis of DNA was activated by bivalent metal ions and also by ATP and NAD. Unlike the main fraction of lactoferrin with the highest affinity for Blue Sepharose, all protein subfractions with DNase activity were cytotoxic and suppressed growth of human and mouse tumor cell lines.

Human deoxyribonucleases.

Although mammalian deoxyribonucleases were discovered more than 60 years ago, interest in these enzymes is not weakening. During the last decade, intensive studies of human DNases culminated in discovery of several novel enzymes exhibiting DNase activity. These include an unusual DNase, lactoferrin. For some enzymes, their three-dimensional structure and molecular mechanisms underlying their functioning have been elucidated. In patients with some autoimmune and viral diseases, catalytic antibodies also contribute to alternative pathways of DNA hydrolysis. Some enzymes exhibiting DNase activity play an important role in pathogenesis of various diseases and also in programmed cell death (apoptosis). This review highlights recent achievement in human deoxyribonuclease research. It also considers mechanisms of DNA hydrolysis. The review also summarizes modern data on the biological role of these enzymes in functioning of the human organism, realization of its protective mechanisms, and possible applications of DNases in medicine.

Catalytic heterogeneity of polyclonal DNA-hydrolyzing antibodies from the sera of patients with multiple sclerosis

Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase activity is associated with IgGs from the sera of patients with multiple sclerosis (MS) but not with those from the sera of normal humans. Here we present evidence showing that MS IgG, its F(ab) fragments, and separated L-chains catalyze DNA hydrolysis. The properties of the DNase activity of these polyclonal IgGs distinguish them from other known human DNases. In addition, their specific activities with different oligonucleotide substrates and the range of optimal pHs, apparent K M values and substrate specificities varied widely for different patients. The findings speak in favor of the generation by the immune systems of individual patients of a variety of polyclonal catalytic IgG pools, from relatively small to extremely large ones.

Molecular Characterization of a Novel Endonuclease (Xib) and Possible Involvement in Lysosomal Glycogen Storage Disorders

We cloned and partially characterized a human endonuclease (Xib) which shows sequence homologies to pancreatic DNase I but an enzymatic activity closer to DNase II. We report on the structural differences found between Xib and other recently cloned human DNases. Fluores cence microscopy analysis of transiently transfected cells with Xib::pEGFP constructs indicate that the protein is located in the cytoplasm and possibly anchored to a membrane, as deduced from a hydrophobic amino acid stretch present at the C-terminal end. Xib is overexpressed in muscle and cardiac tissues and is alternately spliced in several normal and neoplastic cells. In situ hybridization studies using human cardiac and muscle biopsies indicate accumulation of Xib transcript in the vacuoles of muscle cells from patients affected by vacuolar myopathy as acid maltase deficiency; however, no point mutations were detected in their DNA.

Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis.

DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA-cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.